< 19 > F-PDA-DNA-DTPA / Gd nano sensing probe, preparation method thereof and application of < 19 > F-PDA-DNA-DTPA / Gd nano sensing probe in fluorine magnetic resonance imaging

Abstract

The invention provides a < 19 > F-PDA-DNA-DTPA / Gd nano sensing probe, a preparation method thereof and application of the < 19 > F-PDA-DNA-DTPA / Gd nano sensing probe in fluorine magnetic resonance imaging. The nano sensing probe is composed of an MR signal source, a quenching agent and a response connector. Wherein the MR signal source is perfluorocarbon nanoparticles with high-density fluorine, the quenching agent is Gd-DTPA, the response connector is a DNA aptamer and is composed of a DNA single chain S1 and a complementary chain S2 thereof, and the nucleotide sequences of the DNA single chain S1 and the complementary chain S2 are shown as SEQ ID NO. 1 and SEQ ID NO. 2. They are visible only upon activation of a specific biological trigger as a responsive nanoparticle platform, and biological applications thereof are demonstrated by targeting thrombin using aptamers and by exhibiting a strong and selective response to thrombin in live mice.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological probes and specifically relates to a 19 F-PDA-DNA-DTPA / Gd nanosensor probe and its preparation method and application in fluorine magnetic resonance imaging. Background Art

[0002] Magnetic resonance imaging (MRI) is a noninvasive diagnostic technique used to visualize anatomical structures and physiological functions. The development of MRI molecular imaging probes is critical for the diagnosis and prognosis of a variety of adverse health conditions. Among them, the most intensively studied probes are gadolinium (Gd)-based contrast agents. Some contrast agents enhance the relaxation rate of protons in the surrounding water molecules by changing the relaxation rate of protons in the surrounding water molecules. 1 H MRI contrast. Although contrast enhancement 1 H MRI has great application value in molecular imaging, but its low sensitivity and large background signal limit its application.

[0003] Fluorine MRI ( 19 F MRI) is 1 H MRI is an emerging alternative technique that produces low background signals due to its extremely low endogenous fluoride concentration. This enables direct and quantitative detection of 19 The probe of F. 19 F MR images can be compared with anatomical 1 H MR images are superimposed to provide complementary information. Fluorinated nanoparticles as 19 F MRI imaging agents have attracted much attention. They can encapsulate high concentrations of fluorinated molecules in aqueous media. The encapsulation materials used include phospholipids, polymer surfactants, or solid coatings (such as silica). Phospholipid-coated perfluorocarbon nanoemulsions have been used for in vitro cell labeling and in vivo tracking of macrophage uptake. Targeted nanoemulsions have been used to image integrins including ανβ 3 , fibrin clot, and α2-antiplasmin.

[0004] In the field of molecular imaging, of particular interest are “on” sensors, where the signal of the imaging probe is quenched before interaction with a specific analyte and turned on after the interaction. 19 F signal. This type of probe is more analyte-specific than targeted probes, because targeted probes generate signals regardless of whether they bind to the target. In his seminal work, Kikuchi reported several silica-coated perfluorocarbon core-shell nanoparticle “on” probes, in which gadolinium ions (Gd 3+) complexes are coupled to the nanoparticle surface. These probes achieve signal on / off switching through distance-dependent paramagnetic relaxation enhancement; an “on” signal is obtained when the complex is cleaved from the surface. This mechanism has been used to detect reducing environments and a variety of hydrolases, including visualization of caspase-3 / 7 activity in living mice. However, one drawback of these probes is that a single gadolinium ion (Gd 3+ ) complexes were unable to strongly quench the signal of perfluorocarbon nanoparticles, so thousands of complexes were required to quench a single nanoparticle. This limited the sensitivity of the probe because a large number of cleavages needed to occur to achieve detectable signal levels. Summary of the invention

[0005] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0006] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0007] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a 19 F-PDA-DNA-DTPA / Gd nanosensing probe.

[0008] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0009] (i) It is composed of an MR signal source, a quencher, and a response connector;

[0010] (ii) The MR signal source is a high-density fluorine perfluorocarbon nanoparticle, the quencher is Gd-DTPA, and the response linker is a DNA aptamer, which is composed of a DNA single strand S1 and its complementary strand S2, and the nucleotide sequence is shown in SEQ ID NO.1 and SEQ ID NO.2.

[0011] Another object of the present invention is to provide a 19 Preparation method of F-PDA-DNA-DTPA / Gd nanosensor probe.

[0012] In order to solve the above technical problems, the present invention provides the following technical solutions: comprising:

[0013] 100 mg of hexadecyltrimethylammonium bromide was dispersed in 10-15 mL of water and subjected to ultrasonic treatment to obtain a dispersion;

[0014] 55-65 μL of perfluorocarbon was added to the dispersion, followed by ultrasonic treatment at 50°C for 2 h to obtain a nanoemulsion suspension;

[0015] Add 90-110 μL NH 3 ·H 2 O and 25-35 mL of water, then add 1-3 mL of 45-55 mg / mL DA aqueous solution and stir to obtain 19 F-PDA solution;

[0016] 19 F-PDA reacts with DNA single strand S1 in Tris buffer under vigorous stirring, centrifuges and washes, and the obtained product reacts with 8-12 mM DNA complementary strand S2 in PBS buffer and then centrifuges and washes to obtain 19 F-PDA-DNA;

[0017] 8~12mmol DTPA-DA is dissolved in 19 F-PDA-DNA was added to an aqueous solution to obtain a mixed solution, the pH of the mixed solution was adjusted, and DTPA-modified DNA was obtained by centrifugation. 19 F-PDA-DNA nanoprobe;

[0018] DTPA-modified 19 The F-PDA-DNA nanoprobes were washed and dispersed in deionized water to obtain DTPA-modified 19 F-PDA-DNA nanoprobe solution;

[0019] Stir DTPA-modified 19 F-PDA-DNA nanoprobe solution, to which GdCl was added 3 Gd 3+ After complexation, pH adjustment, centrifugation and washing to remove unreacted Gd 3+ , and finally obtain 19 F-PDA-DNA-DTPA / Gd nanoprobe.

[0020] As described in the present invention 19 A preferred embodiment of the method for preparing the F-PDA-DNA-DTPA / Gd nanosensor probe, wherein: the ultrasonic treatment time of the dispersion obtained by ultrasonic treatment is 30 to 60 minutes.

[0021] As described in the present invention 19 A preferred embodiment of the method for preparing the F-PDA-DNA-DTPA / Gd nanosensor probe, wherein: the ultrasonic time for obtaining the nanoemulsion suspension by ultrasonic treatment is 1 to 2 hours, and the ultrasonic temperature is 45 to 55°C.

[0022] As described in the present invention 19 A preferred method for preparing the F-PDA-DNA-DTPA / Gd nanosensor probe, wherein: 19 The reaction time of F-PDA and DNA single strand S1 in Tris buffer under vigorous stirring is 10 to 14 hours.

[0023] As described in the present invention 19 A preferred embodiment of the method for preparing the F-PDA-DNA-DTPA / Gd nanosensor probe, wherein the reaction temperature of the hybridization reaction is 35-40°C.

[0024] As described in the present invention 19 A preferred embodiment of the method for preparing the F-PDA-DNA-DTPA / Gd nanosensor probe, wherein the reaction time of the hybridization reaction is 1.5 to 2.5 hours.

[0025] As described in the present invention 19 A preferred embodiment of the method for preparing the F-PDA-DNA-DTPA / Gd nanosensor probe, wherein: the pH of the mixed solution is adjusted to 6.5-7.5 by using NaOH.

[0026] As described in the present invention 19 A preferred method for preparing F-PDA-DNA-DTPA / Gd nanosensor probes, wherein: the DTPA-modified 19 The stirring time of the F-PDA-DNA nanoprobe solution is 2.5 to 3.5 h.

[0027] As described in the present invention 19 A preferred method for preparing the F-PDA-DNA-DTPA / Gd nanosensor probe, wherein: the pH is adjusted and then centrifuged and washed to remove unreacted Gd 3+ The pH is adjusted to 6-7.

[0028] Beneficial effects of the present invention:

[0029] In summary, the present invention designs a 19 The F-PDA-DNA-DTPA / Gd nanoprobe, which is a responsive nanoparticle platform that is only visible when activated by a specific biological trigger, demonstrated its biological application by targeting thrombin using aptamers and showing a strong and selective response to thrombin in living mice. In addition, the selected aptamer DNA was used for the first time in photothermal melting without the need for an enzyme response. Through photothermal response, the probe self-dissociation can achieve a signal transition from no to present.

[0030] The present invention uses19 Initial demonstration of in vivo sensing by F-PDA-DNA-DTPA / Gd nanoprobes, combined with 19 The fields of FMRI, aptamer sensors, and molecular diagnostics provide insights into the development of diagnostic probes that activate in the presence of important disease-related analytes. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0032] Figure 1 The present invention is based on aptamers 19 F MRI biosensor probe design.

[0033] Figure 2 It is a heat map and electrophoresis diagram of the base pair pairing probability in the DNA aptamer of the present invention.

[0034] Figure 3 For the present invention 19 TEM and elemental spectrum of F-PDA-DNA-DTPA / Gd.

[0035] Figure 4 For the present invention 19 DLS analysis of F-PDA-DNA-DTPA / Gd.

[0036] Figure 5 For the present invention 19 Magnetic resonance imaging evaluation of F-PDA-DNA-DTPA / Gd probe.

[0037] Figure 6 The nanoprobes of different concentrations are 19 Cell viability of cells co-incubated with F-PDA-DNA-DTPA / Gd.

[0038] Figure 7 For the present invention 19 Blood compatibility analysis of F-PDA-DNA-DTPA / Gd at different concentrations.

[0039] Figure 8 For the present invention 19 Blood analysis data of the F-PDA-DNA-DTPA / Gd nanoprobe group and the control group.

[0040] Fig. 9 Injection of the present invention 19Mouse organ tissue sections at different times after F-PDA-DNA-DTPA / Gd. (Scale bar: 100m)

[0041] Fig.10 Injection of the present invention 19 Mice treated in different ways after F-PDA-DNA-DTPA / Gd 1 H / 19 F Magnetic resonance imaging.

[0042] Fig.11 The present invention is co-incubated with different concentrations of thrombin under 480nm excitation. 19 Fluorescence spectrum of DOX released by F-PDA-DNA-DTPA / Gd probe.

[0043] Fig.12 For the present invention 19 Laser confocal images of F-PDA-DNA-DTPA / Gd probes with different treatments. (Scale bar: 10 μm)

[0044] Fig.13 The samples prepared under different DA aqueous solution dosage conditions in Comparative Example 1 of the present invention are 19 Transmission electron microscopy image of F-PDA-DNA-DTPA / Gd nanoprobe. DETAILED DESCRIPTION

[0045] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0046] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0047] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0048] Unless otherwise specified, the raw materials used in the present invention are all commercially available in the art, specifically:

[0049] Hexadecyltrimethylammonium bromide, eicosine-15-crown-5-ether, dopamine hydrochloride (DA, purity 99%), tetraethyl silicate, ethyl acetate, propidium iodide (PI), doxorubicin hydrochloride (DOX, purity 98%) and calcein acetoxymethyl ester (CalceinAM) were purchased from Aladdin, and diethylenetriaminepentaacetic dianhydride (DTPA-DA, 95%) and gadolinium (III) chloride hexahydrate (GdCl3, 99.9%) were purchased from Alfa Aesar.

[0050] All oligonucleotides used in this study were synthesized by Shanghai Sangon Biotechnology Technology Service Co., Ltd. and purified by high-performance liquid chromatography (HPLC) (sequences are shown in Table 1)

[0051] Table 1 Oligonucleotide sequences

[0052]

[0053] The present invention refers to the following method 19 The relevant properties of F-PDA-DNA-DTPA / Gd were evaluated and tested:

[0054] In vitro cytotoxicity assessment

[0055] The cells were added to a 96-well cell plate containing culture medium and incubated at 37°C for 24 hours in an environment containing 5% carbon dioxide and 95% humidity to allow the cells to spread throughout the cell plate. The culture medium was then removed and different concentrations of 19 Incubate the cells in the medium containing F-PDA-DNA-DTPA / Gd for 24 hours (note that the cell density in the 96-well plate must be greater than 70%). After washing twice with PBS, add 10% CCK-8 medium to the cells and culture for another 2 hours. Finally, measure the absorbance at 450nm with an enzyme reader and calculate the cell survival rate using the following formula:

[0056] Cell survival rate (%) = absorbance of experimental well - absorbance of blank well / absorbance of control well - absorbance of blank well × 100%;

[0057] Blood tests

[0058] Two groups (n=5) of healthy mice were anesthetized with isoflurane; one group was injected 19 F-PDA-DNA-DTPA / Gd solution was used for injection, and the other group was not treated. The behavioral changes and weight changes of the mice after injection were observed. One month later, blood samples were collected for blood tests.

[0059] Construction of tumor model

[0060] HepG2 cells were cultured in DMEM medium at 37°C in an environment containing 5% carbon dioxide and 95% humidity. When the cell number reached the desired level, the cells were digested and washed several times with PBS and then dispersed in serum-free DMEM medium. A total of 2 × 10 6 4T1 cells were injected intramuscularly into each BALB / c mouse.

[0061] Example 1

[0062] This embodiment provides a 19 The preparation method of F-PDA-DNA-DTPA / Gd nanoprobe is as follows:

[0063] 1) 19 Synthesis of F-PDA;

[0064] 100 mg of hexadecyltrimethylammonium bromide (CTAB) was dispersed in 12 mL of water and ultrasonically treated for 30 minutes to obtain a dispersion;

[0065] 60 μL of perfluorocarbon compound (PFCE) was added to the dispersion, followed by ultrasonic treatment at 50 °C for 2 h to obtain a nanoemulsion suspension;

[0066] Add 100 μL NH 3 ·H 2 O and 30 mL of water, then 2 mL of 50 mg / mL DA aqueous solution was added and stirred for 24 h to obtain 19 F-PDA.

[0067] 2) 19 Synthesis of F-PDA-DNA;

[0068] 19 F-PDA and DNA single strand S1 were reacted in Tris buffer (10 mM, pH 8.5) under vigorous stirring for 12 h, then centrifuged at 10,000 rpm and washed three times. The resulting product and 10 mM DNA complementary strand S2 were mixed in PBS buffer (NaCl 136.89 mM, KCl 2.67 mM, Na 2 HPO 4 8.1 mM, KH 2 PO 4 1.76 mM, pH 7.4) at 37°C for 2 h, centrifuged at 10,000 rpm and washed three times with deionized water to obtain 19 F-PDA-DNA;

[0069] 3) 19 Synthesis of F-PDA-DNA-DTPA / Gd nanoprobes;

[0070] 10 mmol of DTPA-DA was dissolved in 19 an aqueous solution of F-PDA-DNA to obtain a mixed solution;

[0071] NaOH was added to the mixture to adjust the pH to 7. After stirring for 24 h, the DTPA-modified 19 F-PDA-DNA nanoprobe was obtained by centrifugation, washed 3 times with deionized water, and dispersed in deionized water to obtain a DTPA-modified 19 F-PDA-DNA nanoprobe solution;

[0072] Stirred at room temperature for 3 h, GdCl 3 was added to the DTPA-modified 19 F-PDA-DNA nanoprobe solution for Gd 3+ complexation. NaOH was added thereto to adjust the pH to 6.5, centrifuged at 10,000 rpm and washed 3 times with water to remove unreacted Gd 3+ , and finally 19 F-PDA-DNA-DTPA / Gd nanoprobe was obtained.

[0073] Refer to Figure 1 , for the design diagram of the F-PDA-DNA-DTPA / Gd nanoprobe of the present invention 19 , which is the base pair pairing probability heat map and electrophoresis diagram. Figure 2 In the electrophoresis diagram, lane 1 is the marker, lane 2 is S1, lane 3 is S2, and lane 4 is S1S2. The single-stranded DNA S1 was linked to the surface of pre-synthesized Figure 2 F-PDA and hybridized with its complementary strand DNA S2 ( 19 ). Subsequently, diethylenetriaminepentaacetic dianhydride (DTPA-DA) was conjugated to the 3'-end of DNA S2, and then the DTPA-modified Figure 2 ) The F-PDA-DNA nanoprobe generated the 19 F-PDA-DNA-DTPA / Gd nanoprobe by chelating Gd ions. 19 F-PDA-DNA-DTPA / Gd nanoprobe.

[0074] Figure 3 is 19 the transmission electron microscope (TEM) image of the F-PDA-DNA-DTPA / Gd nanoprobe, and the image shows 19 F-PDA-DNA-DTPA / Gd is a monodisperse spherical nanoprobe.

[0075] Figure 4 is 19DLS analysis of F-PDA-DNA-DTPA / Gd nanoprobes showed that 19 The average particle size of F-PDA-DNA-DTPA / Gd is 110 nm, and the circulation time of the nanoprobe in the body depends on its size and shape, so this property is very ideal for biological and clinical applications.

[0076] Magnetic resonance imaging was used to evaluate 19 The response of F-PDA-DNA-DTPA / Gd to thrombin in vitro is shown in Figure 5 As shown, with the increase of thrombin concentration 19 F and 1 The H imaging signal increased significantly, indicating that the binding of thrombin caused the separation of the complementary DNA strands, with the maximum opening state reached at a thrombin concentration of 4 μM. Initially the signal was barely detectable, but increased significantly after incubation with thrombin.

[0077] In vitro cytotoxicity assessment

[0078] The cells were added to a 96-well cell plate containing culture medium and incubated at 37°C for 24 hours in an environment containing 5% carbon dioxide and 95% humidity to allow the cells to spread throughout the cell plate. The culture medium was then removed and different concentrations of 19 Incubate the cells in the medium containing F-PDA-DNA-DTPA / Gd for 24 hours (note that the cell density in the 96-well plate must be greater than 70%). After washing twice with PBS, add 10% CCK-8 medium to the cells and culture for another 2 hours. Finally, measure the absorbance at 450nm with an enzyme reader. The cell survival rate is calculated by the following formula

[0079] Figure 6 HepG2 cells were treated with different concentrations of 19 Activity data of cells after co-incubation with F-PDA-DNA-DTPA / Gd nanoprobes (CCK-8 test results). As shown in the figure, cells showed high survival rates at all tested concentrations. However, no effect of nanoparticles on cell proliferation and differentiation was observed. Even at the highest concentration (400 μg / mL), at least 90% of the cells survived, which highlights the extremely low toxicity level of the nanoprobes.

[0080] In vitro blood compatibility study is another important indicator for evaluating the toxicity of materials and, to a certain extent, determines whether the material has the possibility of in vivo application. 19The PBS buffer solution of F-PDA-DNA-DTPA / Gd was incubated with red blood cells for 2 hours. It is well known that in water, the intracellular osmotic pressure of red blood cells is different from that in the outside world. Red blood cells will continuously absorb water and cause rupture. The PBS buffer solution is similar to the internal environment of red blood cells, and red blood cells will not absorb water and cause rupture. As shown in 7, we found that it has good blood compatibility. At a concentration of 400μg / mL, red blood cells remained intact, with no significant difference from the negative control group. No ruptured red blood cells were detected in the supernatant. By calculation, the hemolysis rate of red blood cells was extremely low, which indicates that 19 F-PDA-DNA-DTPA / Gd has the potential for in vivo application. Based on this, we further studied 19 Long-term toxicity of F-PDA-DNA-DTPA / Gd to mice. After intravenous injection of the nanomaterial, the mice remained healthy for one month, with a slight increase in weight, and no abnormalities in diet, appearance, activity, exploratory behavior, urination or nervous system, which was no different from the control group.

[0081] Clinical blood routine examination Figure 8 , the experimental group showed normal results compared with the control group.

[0082] The results of organ tissue sections stained with hematoxylin and eosin (H&E) are as follows Fig. 9 , the results show 19 F-PDA-DNA-DTPA / Gd does not cause damage or inflammation to the heart, liver, spleen, lungs, kidneys and other organs, ensuring the good biocompatibility of the nanoprobe in the body during diagnosis and treatment. Based on these results, it can be concluded that 19 F-PDA-DNA-DTPA / Gd has no obvious adverse reactions in vivo and has good biocompatibility, indicating that the probe can be used in vivo.

[0083] pass 19 F MRI test 19 The response effect of F-PDA-DNA-DTPA / Gd in living mice, specifically, 19 The F-PDA-DNA-DTPA / Gd probe was injected into the tumor of Balb / c mice, followed by injection of PBS (control group) and thrombin. Fig.10 As shown, 19 F MR images showed a bright and distinct signal at the site of thrombin injection, but no signal at the site of PBS injection. 1 H MRI also has obvious signal enhancement. 19 The response of F-PDA-DNA-DTPA / Gd to thrombin was clear and distinct from the response in the absence of target.

[0084] Embedding the chemotherapy drug doxorubicin (DOX) 19 In F-PDA-DNA-DTPA / Gd, the -CG- bases of the DNA double strands provide loading sites for DOX. 19 After centrifugation of F-PDA-DNA-DTPA / Gd, the fluorescence spectrum was measured, and there was no fluorescence intensity, indicating that DOX was not released. However, after centrifugation of PDA-DNA-DTPA / Gd co-incubated with thrombin, the fluorescence spectrum was measured, and there was a certain fluorescence intensity, indicating that DOX was released after laser irradiation.

[0085] Then, the supernatant of centrifugation after co-incubation with different concentrations of thrombin was taken to measure the fluorescence spectrum, such as Fig.11 As shown in the figure, the fluorescence intensity gradually increased with the increase of thrombin concentration, indicating that 19F-PDA-DNA-DTPA / Gd can release more DOX with the increase of thrombin concentration. Fig.12 It is shown that no fluorescence is generated when the cells are co-incubated with PBS, indicating that the DNA double strands are not unwound and DOX is not released. However, red fluorescence is generated after co-incubation with thrombin, indicating that thrombin specifically binds to the aptamer, releasing DOX and generating a fluorescent signal.

[0086] Comparative Example 1

[0087] The difference between this comparative example and Example 1 is that the volume of 50 mg / mL DA aqueous solution added to the nanoemulsion suspension in step 1) is adjusted to 0.5 mL, 1 mL, and 1.5 mL respectively, and the remaining steps and processes are all referred to Example 1, so as to obtain different DA aqueous solution dosages of this comparative example. 19 F-PDA-DNA-DTPA / Gd nanoprobe.

[0088] Fig.13 The dosage of different DA aqueous solutions in this comparative example 19 It can be seen from the F-PDA-DNA-DTPA / Gd nanoprobe that the probe morphology under this condition is not uniform, while Example 1 of the present invention can form a monodisperse uniform nanoprobe under the condition of 2 ml dosage.

[0089] In summary, the present invention designs a 19The F-PDA-DNA-DTPA / Gd nanoprobe, which is a responsive nanoparticle platform that is only visible when activated by a specific biological trigger, demonstrated its biological application by targeting thrombin using aptamers and showing a strong and selective response to thrombin in living mice. In addition, the selected aptamer DNA was used for the first time in photothermal melting without the need for an enzyme response. Through photothermal response, the probe self-dissociation can achieve a signal transition from no to present.

[0090] The present invention uses 19 Initial demonstration of in vivo sensing by F-PDA-DNA-DTPA / Gd nanoprobes, combined with 19 The fields of FMRI, aptamer sensors, and molecular diagnostics provide insights into the development of diagnostic probes that activate in the presence of important disease-related analytes.

[0091] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A 19 F-PDA-DNA-DTPA / Gd nanosensor probe, characterized by: (i) It is composed of an MR signal source, a quencher, and a response connector; (ii) The MR signal source is a high-density fluorine perfluorocarbon nanoparticle, the quencher is Gd-DTPA, and the response linker is a DNA aptamer, which is composed of a DNA single strand S1 and its complementary strand S2, and the nucleotide sequence is shown in SEQ ID NO.1 and SEQ ID NO.

2.

2. A 19 The preparation method of F-PDA-DNA-DTPA / Gd nanoprobe is characterized by: The method comprises dispersing 100 mg of hexadecyltrimethylammonium bromide in 10 to 15 mL of water and subjecting the mixture to ultrasonic treatment to obtain a dispersion; 55-65 μL of perfluorocarbon was added to the dispersion, followed by ultrasonic treatment at 50°C for 2 h to obtain a nanoemulsion suspension; Add 90-110 μL NH 3·H 2 O and 25-35 mL water to the nanoemulsion suspension, then add 1-3 mL of 45-55 mg / mL DA aqueous solution and stir to obtain 19 F-PDA solution; 19 F-PDA reacts with DNA single strand S1 in Tris buffer under vigorous stirring, centrifuges and washes, and the obtained product reacts with 8-12 mM DNA complementary strand S2 in PBS buffer and then centrifuges and washes to obtain 19 F-PDA-DNA; 8~12mmol DTPA-DA is dissolved in 19 F-PDA-DNA was added to an aqueous solution to obtain a mixed solution, the pH of the mixed solution was adjusted, and DTPA-modified DNA was obtained by centrifugation. 19 F-PDA-DNA nanoprobe; DTPA-modified 19 The F-PDA-DNA nanoprobes were washed and dispersed in deionized water to obtain DTPA-modified 19 F-PDA-DNA nanoprobe solution; Stir DTPA-modified 19 F-PDA-DNA nanoprobe solution, and GdCl3 was added to it for Gd 3+ After complexation, pH adjustment, centrifugation and washing to remove unreacted Gd 3+ , and finally obtain 19 F-PDA-DNA-DTPA / Gd nanoprobe.

3. The method according to claim 2 19 The preparation method of F-PDA-DNA-DTPA / Gd nanoprobe is characterized by: The ultrasonic treatment time of the dispersion obtained by the ultrasonic treatment is 30 to 60 minutes.

4. The method according to claim 2 19 The preparation method of F-PDA-DNA-DTPA / Gd nanoprobe is characterized by: The ultrasonic treatment to obtain the nanoemulsion suspension has an ultrasonic time of 1 to 2 hours and an ultrasonic temperature of 45 to 55°C.

5. The method according to claim 2 19 The preparation method of F-PDA-DNA-DTPA / Gd nanoprobe is characterized by: Said 19 The reaction time of F-PDA and DNA single strand S1 in Tris buffer under vigorous stirring is 10 to 14 hours.

6. The method according to claim 2 19 The preparation method of F-PDA-DNA-DTPA / Gd nanoprobe is characterized by: The reaction temperature of the hybridization reaction is 35-40°C.

7. The method according to claim 6 19 The preparation method of F-PDA-DNA-DTPA / Gd nanoprobe is characterized by: The reaction time of the hybridization reaction is 1.5 to 2.5 hours.

8. The method according to claim 2 19 The preparation method of F-PDA-DNA-DTPA / Gd nanoprobe is characterized by: The pH of the mixed solution is adjusted to 6.5-7.5 by using NaOH.

9. The method according to claim 2 19 The preparation method of F-PDA-DNA-DTPA / Gd nanoprobe is characterized by: The DTPA-modified 19 The stirring time of the F-PDA-DNA nanoprobe solution is 2.5 to 3.5 h.

10. The method according to claim 2 19 The preparation method of F-PDA-DNA-DTPA / Gd nanoprobe is characterized by: After adjusting the pH, centrifuge and wash to remove unreacted Gd 3+ The pH is adjusted to 6-7.

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